title: Differences in purinergic amplification of osmotic cell lysis by the pore-forming RTX toxins Bordetella pertussis CyaA and Actinobacillus pleuropneumoniae ApxIA: the role of pore size.
abstract: A large subgroup of the repeat in toxin (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae, which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis, which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity (>20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins.
authors.affiliation: Institute of Microbiology of the ASCR, v.v.i., Prague, Czech Republic.
authors.forename: Jiri
authors.initials: J
authors.lastname: Masin
authors.forename: Radovan
authors.initials: R
authors.lastname: Fiser
authors.forename: Irena
authors.initials: I
authors.lastname: Linhartova
authors.forename: Radim
authors.initials: R
authors.lastname: Osicka
authors.forename: Ladislav
authors.initials: L
authors.lastname: Bumba
authors.forename: Erik L
authors.initials: EL
authors.lastname: Hewlett
authors.forename: Roland
authors.initials: R
authors.lastname: Benz
authors.forename: Peter
authors.initials: P
authors.lastname: Sebo
mesh_terms: D016977:Actinobacillus pleuropneumoniae / Q000378:metabolism*; D037361:Adenylate Cyclase Toxin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D001886:Bordetella pertussis / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004912:Erythrocytes / Q000378:metabolism; D006460:Hemolysin Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006461:Hemolysis; D006593:Hexokinase; D008051:Lipid Bilayers / Q000378:metabolism; D008264:Macrophages; D051379:Mice; D009997:Osmotic Pressure; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives; D012394:Rosaniline Dyes
chemical_list: D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; D012394:Rosaniline Dyes; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; C087744:ApxI toxin, Bacteria; D011732:Pyridoxal Phosphate; D006593:Hexokinase; C004692:coomassie Brilliant Blue